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Biomedical subjects

L Zhu

Publications and source records attributed to L Zhu.

At least 199 records · Page 11Linked to original sources
PubMed1999

[Cloning and sequence analysis of 55 K protein of egg drop syndrome virus].

OBJECTIVE: For understanding the characteristics of genomic structure of egg drop syndrome virus(EDSV). METHODS: Nucleic acid was extracted using routine method from weak virulent strain AA-2 of EDSV isolated from Chinese sick hens. Construction of the whole genomic library was by hydrolysis with Hind III, strand encoding 55 K gene locating in Hind III--A segment was sequenced and analyzed. RESULTS: The open reading frame has a length of 1,014 nt and codes a polypeptide of 337 amino acids with molecular weight of 38,200. Analysis of the amino acid sequence revealed a homology from 25.5%-32.4% to the 55 K protein of human adenovirus types 2, 12, 40, canine adenovirus and fowl adenoviruses of group 1, whereas to ovine adenovirus is 46.4%. CONCLUSION: The genomic structure of EDSV has some relationship with adenoviruses.

Animals↗
PubMed1999

Characterization of a 3'-5' exonuclease associated with VDJP.

VDJP (V(D)J RSS Dependent DNA Joining Protein) was cloned based on binding to the nonamer portion of the V(D)J recombinational signal sequence (RSS), and genetic analysis revealed that VDJP is encoded by the same gene as the large subunit of Replication Factor C (RF-C). Recombinant VDJP has a site directed DNA joining activity and is capable of forming a covalent bond between DNA fragments containing an RSS element near their ends and exhibits 3' to 5' exonuclease activity. In this report, we examine the biochemical properties of the VDJP exonuclease activity such as directionality of nuclease action (3' to 5' or 5' to 3'), single-strand substrate preference, cleavage products, dependence on cofactors and metal cations, and optimal reaction conditions. From this analysis, we conclude that VDJP has an intrinsic 3'-5' exonuclease activity that produces mononucleotide products.

DNA↗
PubMed1999

DP1 phosphorylation in multimeric complexes: weaker interaction with cyclin A through the E2F1 cyclin A binding domain leads to more efficient phosphorylation than stronger interaction through the p107 cyclin A binding domain.

Stable enzyme-substrate interaction has been recognized as a major mechanism underlying the substrate preferences of cyclin-dependent kinases (Cdks). To learn the relationship between stability of physical association and efficiency of phosphorylation, we studied DP1 phosphorylation by cyclin A-Cdk2 in multiprotein complexes. When DP1 was connected to cyclin A-Cdk2 through E2F4 and p107, its phosphorylation was very inefficient, although its association with cyclin A-Cdk2 was stable. In contrast, DP1 was efficiently phosphorylated when weakly connected to cyclin A-Cdk2 via E2F1 or E2F4 with a fused cyclin A binding domain of E2F1. The transactivation activity of E2F4-DP1 heterodimers was reduced when DP1 was phosphorylated, while a phosphorylation deficient mutant of DP1 resisted this down-regulation. Phosphorylation and functional regulation of DP1 were not due to nuclear localization. Thus, stronger physical association between the kinase and the substrate does not necessarily lead to more efficient phosphorylation than weaker interaction does.

Amino Acid Sequence↗
PubMed1999

Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells.

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.

Adolescent↗
PubMed1999

Identification of the block in targeted retroviral-mediated gene transfer.

A chimeric retroviral vector (33E67) containing a CD33-specific single-chain antibody was generated in an attempt to target cells displaying the CD33 surface antigen. The chimeric envelope protein was translated, processed, and incorporated into viral particles as efficiently as wild-type envelope protein. The viral particles carrying the 33E67 envelope protein could bind efficiently to the CD33 receptor on target cells and were internalized, but no gene transfer occurred. A unique experimental approach was used to examine the basis for this postbinding block. Our data indicate that the chimeric envelope protein itself cannot participate in the fusion process, the most reasonable explanation being that this chimeric protein cannot undergo the appropriate conformational change that is thought to be triggered by receptor binding, a suggested prerequisite to subsequent fusion and core entry. These results indicate that the block to gene transfer in this system, and probably in most of the current chimeric retroviral vectors to date, is the inability of the chimeric envelope protein to undergo this obligatory conformational change.

3T3 Cells↗
PubMed1999

Primary structure, tissue distribution, and expression of mouse phosphoinositide-dependent protein kinase-1, a protein kinase that phosphorylates and activates protein kinase Czeta.

Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified serine/threonine kinase that phosphorylates and activates Akt and p70(S6K), two downstream kinases of phosphatidylinositol 3-kinase. To further study the potential role of PDK1, we have screened a mouse liver cDNA library and identified a cDNA encoding the enzyme. The predicted mouse PDK1 (mPDK1) protein contained 559 amino acids and a COOH-terminal pleckstrin homology domain. A 7-kilobase mPDK1 mRNA was broadly expressed in mouse tissues and in embryonic cells. In the testis, a high level expression of a tissue-specific 2-kilobase transcript was also detected. Anti-mPDK1 antibody recognized multiple proteins in mouse tissues with molecular masses ranging from 60 to 180 kDa. mPDK1 phosphorylated the conserved threonine residue (Thr402) in the activation loop of protein kinase C-zeta and activated the enzyme in vitro and in cells. Our findings suggest that there may be different isoforms of mPDK1 and that the protein is an upstream kinase that activates divergent pathways downstream of phosphatidylinositol 3-kinase.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1999

Dynamics of palmitic acid complexed with rat intestinal fatty acid binding protein.

Dynamics of palmitic acid (PA), isotopically enriched with 13C at the second, seventh, or terminal methyl position, were investigated by 13C NMR. Relaxation measurements were made on PA bound to recombinant rat intestinal fatty acid binding protein (I-FABP) at pH 5.5 and 23 degreesC, and, for comparison, on PA incorporated into 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) micelles, and dissolved in methanol. The 13C relaxation data, T1, and steady-state nuclear Overhauser effect (NOE) obtained at two different magnetic fields were interpreted using the model-free approach [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall rotational correlation time of the fatty acid.protein complex was 2.5 +/- 0.4 ns, which is substantially less than the value expected for the protein itself (>6 ns). Order parameters (S2), which are a measure of the amplitude of the internal motion of individual C-H vectors with respect to the PA molecule, while largest for C-2 and smallest for the methyl carbon, were relatively small (<0.4) in the protein complex. S2 values for given C-H vectors also were smaller for PA in the MPPC micelles and in methanol than in the protein complex. Correlation times reflective of the time scale of the internal motion of the C-H vectors were in all cases <60 ps. These results support the view that the fatty acid is not rigidly anchored within the I-FABP binding pocket, but rather has considerable freedom to move within the pocket.

Animals↗
PubMed1999

Understanding the influence of temperature change and cosolvent addition on conversion rate of enzymatic suspension reactions based on regime analysis.

It is a commonly held belief that enzymatic conversions of substrate in aqueous suspensions can be speeded up by raising the temperature or adding organic solvents to promote dissolution of the substrate. To quantify the impact of such changes, we studied the alpha-chymotrypsin-catalyzed hydrolysis of dimethyl benzylmethylmalonate as a model system. It was found that, upon addition of organic cosolvents, longer process times were actually required, even though the substrate solubility increased severalfold as expected. Upon raising the temperature from 25 degrees C to 37 degrees C, on the other hand, both the substrate solubility, the substrate dissolution rate, and the enzymatic reaction rate increased, leading to shorter process times. A dissolution-reaction model incorporating the kinetics of enzyme deactivation could be developed. A simple relation for the prediction of the overall process time was established by evaluating the time constants for the subprocesses: substrate dissolution; enzymatic conversion; and enzyme deactivation. Using regime analysis, rules of thumb for the optimization of an enzymatic suspension reaction were derived.

Animals↗
PubMed1999

IL-10 gene knockout mice show enhanced Th1-like protective immunity and absent granuloma formation following Chlamydia trachomatis lung infection.

We previously reported that higher IL-10 production is correlated with lower IFN-gamma production, weaker delayed hypersensitivity (DTH), and slower organism clearance following chlamydial infection in mice. To assess more directly the role of IL-10, we examined protective immunity and pathological reaction in C57BL/6 IL-10 gene knockout (KO) and wild-type mice. The results showed that in the absence of endogenous IL-10, mice had significantly accelerated chlamydial clearance and developed significantly stronger DTH responses, which could be inhibited by local delivery of rIL-10. Consistent with the enhancement of DTH responses, IL-10 KO mice showed stronger and more persistent CD4 T cell-dependent IFN-gamma production and significant elevation of IL-12 and TNF-alpha production. Additionally, wild-type, but not IL-10 KO, mice showed granuloma formation that was correlated with higher levels of Th2 cytokine (IL-5) production at the later stages of infection. Moreover, chlamydial infection, unlike parasitic protozoan infection, did not induce significant acute toxicity in IL-10 KO mice, which may be due to the low (undetectable) levels of systemic release of proinflammatory cytokines. These results suggest that IL-10 inhibits the priming and expansion of Th1-like T cell responses and that IL-10 plays a role in the fibrotic reaction seen with chlamydial infection.

Animals↗
PubMed1999

Carboxy-substituted cinnamides: a novel series of potent, orally active LTB4 receptor antagonists.

A series of carboxy-substituted cinnamides were investigated as antagonists of the human cell surface leukotriene B4 (LTB4) receptor. Binding was determined through measurement of [3H]LTB4 displacement from human neutrophils. Receptor antagonism was confirmed through a functional assay, which measures inhibition of Ca2+ release in human neutrophils. Potent antagonists were discovered through optimization of a random screening hit, a p-(alpha-methylbenzyloxy)cinnamide, having low-micromolar activity. Substantial improvement of in vitro potency was realized by the attachment of a carboxylic acid moiety to the cinnamide phenyl ring through a flexible tether, leading to identification of compounds with low-nanomolar potency. Modification of the benzyloxy substituent, either through ortho-substitution on the benzyloxy phenyl group or through replacement of the ether oxygen with a methylene or sulfur atom, produced achiral antagonists of equal or greater potency. The most potent compounds in vitro were assayed for oral activity using the arachidonic acid-induced mouse ear edema model of inflammation. Several compounds in this series were found to significantly inhibit edema formation and myeloperoxidase activity in this model up to 17 h after oral administration. Representatives of this series have been shown to be potent and long-acting orally active inhibitors of the LTB4 receptor.

Administration, Oral↗
PubMed1999

Occurrence and progression of dementia in a community population aged 75 years and older: relationship of antihypertensive medication use.

OBJECTIVE: To examine whether antihypertensive medication use can affect the occurrence and progression of dementia. SUBJECTS AND METHODS: In a community cohort of 1810 persons aged 75 years and older, 225 prevalent cases of dementia were detected. Among the 1301 persons without dementia, 224 incident cases of dementia were identified during an average period of 3 years. Among the 225 prevalent cases of dementia, 79 were suitable for the analysis of cognitive decline. Information on drug use was collected for the 2 weeks preceding the baseline interview. RESULTS: Subjects taking antihypertensive medication (n = 651, 83.9% of whom took diuretics) had a lower prevalence of dementia than those not taking antihypertensive medication (P<.001). Subjects without dementia who were taking antihypertensive medication at baseline (n = 584) had a reduced incidence of dementia (adjusted relative risk, 0.7; 95% confidence interval, 0.6-1.0; P = .03). Furthermore, subjects taking diuretics (n = 484) had an adjusted relative risk of 0.7 (95% confidence interval, 0.5-1.0; P = .02) for all dementia, and subjects taking diuretic monotherapy (n = 345) had an adjusted relative risk of 0.6 (95% confidence interval, 0.4-0.9; P = .006). The use of other antihypertensive medication (calcium antagonists or beta-blockers), however, was related to a reduced risk of Alzheimer disease (adjusted relative risk, 0.6; 95% confidence interval, 0.3-1.2) only in the subpopulation with a higher baseline blood pressure (n = 458). Patients with dementia at baseline who were not taking diuretics had a 2-fold faster rate of decline in the score on the Mini-Mental State Examination than those taking diuretics. CONCLUSION: The use of diuretics may protect against dementia in elderly persons.

Aged↗
PubMed1999

Interleukin-2 receptoralpha gene expression in intestinal allograft rejection.

This study was undertaken to determine whether the interleukin-2 receptor alpha (IL-2Ralpha) coding gene could serve as a genetic marker of intestinal allograft rejection. The small bowel graft from Jinbai mice was heterotopically transplanted to BALB/c mice. Naive BALB/c mice and BALB/c mice with intestinal isografts were used as controls. The mRNA transcription level of the spleen cells harvested from the recipients and controls was measured with quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) on postoperative day (POD) 3 and routine pathology was studied in these grafts. The IL-2Ralpha mRNA transcription level in untreated allografts was significantly higher than that of controls on POD 3, when intestinal allograft rejection was not detected. These data suggest that the IL-2Ralpha mRNA transcription level may reflect the early cellular immune response and this genetic marker may be of value in monitoring intestinal allograft rejection.

Animals↗
PubMed1999

Intragraft CD45 RO gene expression is an early marker to detect small bowel allograft rejection in rats.

Wistar Furth (WF) intestinal allografts were transplanted into Sprague-Dawley (SD) rats. Recipients were randomly allocated into the following groups: (1) no treatment; (2) cyclosporine (CsA) 6 mg/kg/day, daily, subcutaneously (s.c.; full-dose therapy); (3) CsA 3 mg/kg/day, daily, s.c. (half-dose therapy); and (4) CsA 3 mg/kg/day, daily, s.c. + Tripterygium Wilfordii Hook. WF (TW) 3 mg/kg/day, daily, s.c. WF rats with intestinal autografts were used as controls. CD45RO intragraft expression and its index (CD45RO/CD45), measured by reverse transcription polymerase chain reaction (RT-PCR), were significantly elevated in untreated and half-dose CsA-treated allografts as early as postoperative day (POD) 4, when rejection of intestinal allografts was not detected by routine pathology. Intestinal permeability measured by Tc-DTPA radioassay was significantly elevated in untreated allografts on POD 6. Histology showed that there was severe rejection in untreated intestinal allografts and mild rejection in allografts treated with a half dose of CsA on POD 6. There was a normal CD45RO expression, permeability, and histology in intestinal allografts treated with either a full dose of CsA or a half dose of CsA + TW. These data indicate that CD45RO intragraft gene expression is an early marker to detect intestinal allograft rejection in rats.

Animals↗
PubMed1999

Rapid colorimetric assay of urinary beta-galactosidase and N-acetyl-beta-D-glucosaminidase with Cobas Mire Auto-analyzer.

A rapid and simple colorimetric method for the assay of urinary beta-galactosidase (GAL) and N-acetyl-beta-D-glucosaminidase (NAG) using 4-nitrophenyl-glycosides as the substrates and a Cobas Mire Auto-analyzer is described. Optimal conditions for this method including substrate concentration, ionic strength, pH and incubation time were characterized. Endogenous substances in urine did not interfere with the assays. Incubation time could be shortened to 10 minutes. A small volume of an alkaline buffer to terminate the enzyme reaction achieved better sensitivity and accuracy. Buffer-substrate solutions were stable for at least one week at 4 degrees C. Normal values of the two urinary enzymes are also reported.

Acetylglucosaminidase↗
PubMed1999

Role of thyroid hormone in stimulating liver repopulation in the rat by transplanted hepatocytes.

Recently, we reported near-complete repopulation of the rat liver by transplanted hepatocytes using retrorsine (RS), a pyrrolizidine alkaloid that alkylates cellular DNA and blocks proliferation of resident hepatocytes, followed by transplantation of normal hepatocytes in conjunction with two-thirds partial hepatectomy (PH). Because two-thirds PH is not feasible for use in humans, in the present study, we evaluated the ability of thyroid hormone (triiodothyronine [T(3)]), a known hepatic mitogen, to stimulate liver repopulation in the retrorsine model. Because T(3) initiates morphogenesis in amphibians through a process involving both cell proliferation and apoptosis, we also determined whether apoptosis might play a role in the mechanism of hepatocyte proliferation induced by T(3). Following hepatocyte transplantation and repeated injections of T(3), the number of transplanted hepatocytes in the liver of RS-pretreated animals increased progressively to repopulate 60% to 80% of parenchymal cell mass in 60 days. We show further that T(3) treatment augments proliferation of normal hepatocytes, as evidenced by increased histone 3 mRNA and cyclin-dependent kinase 2 (cdk2) expression, and this is followed by apoptosis. These combined effects of T(3) lead to selective proliferation of transplanted hepatocytes in RS-pretreated rats, while endogenous hepatocytes, which are blocked in their proliferative capacity by RS, mainly undergo apoptosis. Thus, T(3) can replace PH in the RS-based rat liver repopulation model and therefore represents a significant advance in developing methods for hepatocyte transplantation.

Animals↗
PubMed1999

Macrophages: important accessory cells for reproductive function.

Macrophages are found throughout reproductive tissues. To determine their role(s), we have studied mice homozygous for a null mutation (Csfm(op)) in the gene encoding the major macrophage growth factor, colony-stimulating factor-1 (CSF-1). Both male and female Csfm(op)/Csfm(op) mice have fertility defects. Males have low sperm number and libido as a consequence of dramatically reduced circulating testosterone. Females have extended estrous cycles and poor ovulation rates. CSF-1 is the principal growth factor regulating macrophage populations in the testis, male accessory glands, ovary, and uterus. However, analyses of CSF-1 nullizygous mice suggest that the primary reproductive defect is in the development of feedback regulation of the hypothalamic-pituitary axis. Although not correlating with deficiencies of microglia populations, electrophysiological investigations indicate an impairment of neuronal responses. This suggests that microglia, under the influence of CSF-1, act to organize neuronal connectivity during development and that the absence of this function results in a perturbation of the hypothalamic-pituitary-gonadal axis. Macrophages also appear to have functions in the differentiated tissues of the reproductive system, including having a positive influence on steroidogenic cells. These data suggest that macrophages, through their trophic functions, can be considered as essential accessory cells for normal reproductive functioning.

Animals↗
PubMed1999

Bombesin inhibits apoptosis in developing fetal rat lung.

We have shown recently that apoptosis occurs during fetal and postnatal lung development. Our hypothesis that branching morphogenesis occurs through a delicate balance of cell proliferation and apoptosis predicts that substances that enhance branching of the airways would affect both cell proliferation and apoptosis in the lung. Bombesin-like peptides have a mitogenic effect on bronchial epithelium and fibroblasts, and bombesin has been shown to enhance branching morphogenesis in fetal lung. We used organ cultures of 16-day gestation fetal rat lung to study the effects of bombesin on apoptosis. Cultures were incubated in serumless medium alone or exposed to 1microM bombesin for 0-48 h. Levels of apoptosis were quantified using the TUNEL assay and expressed as percentage of apoptotic cells in paraffin sections of explants. Bombesin significantly inhibited apoptosis in fetal lung mesenchyme 48 h in culture by more than 50% (p < 0.05). The effects of bombesin on apoptosis were prevented completely if explants were exposed to the specific bombesin receptor antagonist, [D-Phe12]bombesin. To examine if the absence of serum in the media could have accounted for some of these effects, explants were cultured for 48 h in serumless medium, medium containing 10% fetal bovine serum, serumless medium with 1 microM bombesin, or medium containing both 10% fetal bovine serum and 1 microM bombesin. The addition of fetal bovine serum to the media reduced apoptosis significantly. The effect of fetal bovine serum on apoptosis was additive with bombesin. We conclude that bombesin inhibits apoptosis in developing fetal rat lung mesenchyme through its interaction with the bombesin receptor.

Animals↗